Development of orthogonal combinations of artificial DNA methyltrasferases and their substrates for study of heritable patterns of DNA methylation.

DNA的共价修饰，如胞嘧啶甲基化，可以诱导可遗传的基因沉默。如果表观遗传修饰可以有针对性，转录治疗的新方法应该会产生。为了应对这一挑战，我们构建了甲基转移酶，通过调整序列使能的组装策略，只在期望的位置起作用。这是序列使能酶重组方法在体内首次成功应用。在本研究中，为了确定分裂的蛋白结构域在DNA结合和甲基化中的功能，我们分别表达和纯化了分裂的蛋白结构域。利用这些结构域，进行DNA结合分析。结果表明，这些结构域协同结合到特定的DNA靶标上。结构域之间的这种相互作用显示了在分裂结构域的靶序列上组装的直接证据。互补蛋白分析已被证明在剖析哺乳动物细胞中的蛋白质相互作用方面是有用的。然而，一些直接的方法已经被用来评价分裂结构域相互作用的动力学。此外，为了扩大基因组DNA上的靶向DNA序列，我们构建了几个锌指结构域。这些结构域显示DNA与内源性靶标结合。为了在哺乳动物细胞中进行有效的DNA甲基化，甲基转移酶的表达将是一个关键因素。从而优化了甲基转移酶的密码子使用。甲基转移酶的表达明显增强。本研究结果将为分离蛋白结构域的设计提供更多的知识。

Covalent modification of DNA, such as cytosine methylation, can induce heritable gene silencing. If epigenetic modifications can be specifically targeted, new approaches to transcriptional therapy should result. To address this challenge we have constructed methyltransferases that would act only at a desired site by adapting the sequence-enabled assembly strategy. This is the first successful application of the sequence-enable enzyme reassembly approach in vivo. In this study, to determine the functions of split protein domains in DNA binding and methylation, the split domains were expressed and purified separately. Utilizing these domains, DNA binding analyses were performed. The results indicate cooperative binding of the domains to the specific DNA targets. This interaction between the domains shows a direct evidence of assembly on the target sequence of split domains. The complementary protein assays have been shown their usefulness in dissection of protein interaction in mammalian cells. However, a few of direct approach to evaluate kinetics of interaction of split domains have been performed. Moreover, to expand the targetable DNA sequences on genomic DNA, several zinc finger domains were constructed. These domains showed DNA binding on endogenous targets. To perform efficient DNA methylation in mammalian cells, the expression of methyltransferase would be a key factor. Thus, the codon usage of methyltransferase was optimized. The expression of methyltransferase was greatly increased. The present results would expand the knowledge in the design of split protein domains.